IS 13829 : 3 993 ( Reaffirmed 2003 ) Indian Standard PESTICIDE - METHOD FOR DETERMINATION OF RESIDUES IN AGRICULTURAL AND FOOD COMMODITIES, SOIL AND WATER ATRAZINE AND SIMAZINE UDC 664 : 543 [ 632'95'028 ATR ] + @ BIS 1993 BUREAU MANAK OF BHAVAN, INDIAN STANDARDS ZAFAR MARC Price Group 2 9 BAHADUR SHAH NEW DELHI 110002 October 1993 Pesticides Residue Analysis Sectional Committee, FAD 34 . FOREWORD This Indian Standard was adopted by the Bureau of Indian Standards after the draft finalized by the Pesticides Residue Analysis Sectional Committee had been approved by the Food and Agriculture Division Council. Both, atrazine ( 2-chloro-4-ethylamino-6-isopropylamino-5-triazine ) and simazine ( 2-chloro-4, 6-bis-ethylamino-s-triazine ) are used as herbicides in agriculture for the control of weeds. Assessment of their residues in food commodities is therefore an important step in safeguarding human health and establishment of regulatory policy. This standard will enable the health authorities and others engaged in the field to follow uniform test procedures for the estimation of residues of atrazine and simazine in various commodities. In the preparation of this standard, due consideration has been given to the maximum limits of atrazine and simazine residues laid under the provisions of Prevention of Food Adulteration Act, The test method is restricted to the prescribed level of 1954 and the Rules framed thereunder. residues. In reporting the results of a test or analysis made in accordance with this standard, if the final value, observed or calculated, is to be rounded off, it shall be done in accordance w$h IS 2 : 1960 `Rules. for rounding off numerical values ( revised )`. IS 13829 : 1993 Indian Standard PESTICIDE-METHODFORDETERMINATION OFRESIDUESINAGRICULTURAEANDFOOD COMMODITIES,SOILANDWATERATRAZINEAND SIMAZINE 1 SCOPE 1.1 This standard describes gas chromatographic ( GLC ) and high performance liquid chromatographic ( HPLC ) methods for determination of atrazine and simazine residues in food commodities. 1.2 The limit of determination of both the compounds is 0'05 pg/g by the PHLC method, whereas it is 0'01 pg/g by the GLC method. 1.3 Though no set procedure for thin layer chromatography ( TLC ) is being prescribed, may be standardized ( TLC ) procedures followed, if necessary, for the purpose of clean up, identification and confirmation of residues of atrazine and simazine. 2 REFERENCES The Indian Standards listed below are necessary adjuncts to this standard: IS No. 1070 : 1992 11380: Title 5.3 Chromatographic i.d. Column, 25 cm long X 2 cm 5.4 Rotary Vacuum Evaporator 5.5 Air Blower 5.6 Water Bath 5.7 Gas Chromatograph Equipped with a nitrogen specific detector and operating under the following suggested psrameters. These parameters may be varied provided according to the available facilitiq, standardization is done: Column : Glass, 100 cm length, 4 mm internal diameter packed with 5 percent OV-101 on Gaschrom Q ( 60-80 ) mesh Temperatures : Column oven : 170°C Injector : 200°C Detector : 210°C Carrier gas ( nitrogen ) flow : 30mI/min rate Retention time : Simazine : 3 minutes approximately Atrazine : 3'5 minutes approximately 5.8 Microlitre Syringe 10 ~1 capacity. Reagent revision ) grade water ( third 1985 Method of sampling for the of pesticide determination residues in agricultural and food commodities OF REAGENTS 3. QUALITY 5.9 High Performance Liquid Chromatograph Unless specified otherwise, pure chemicals and be distilled water ( see IS 1070 : 1992 ) shall employed in the tests. shall mean NOTE - `Pure chemicals' that do not contain impurities which result of analysis. chemicals affect the Equipped with a variable wave length ultraviolet detector, operating under the following suggested parameters. These parameters may be varied according to the available facilities provided standardization is: Column : Zorbax 4 SAMPLING The representative samples for the purpose of estimating residues of atrazine and simazine in the commodities shall be drawn in accordance with IS 11380 : 1985. 5 APPARATUS 5.1 Waring Blender Mobile phase Flow rate ODS or Partisil ODS 25 ~cm length and 4'6 mm internal diameter : Methanol . * 1 ml/min : 230 nm Wave length Absorbance range : 0'02 : Simazine Retention time : 6 minutes approximately Atrazine : 8 minutes .5.2 Laboratory Shaker ( Rotary Action ) 1 approximately IS 13829 : 1993 6 REAGENTS 6.1 Chloroform 6.2 Petroleum PIPLC grade boiling range 40-60°C. ( v/v ) in HPLC grade. carbon . Blend the mixture for 5 minutes and continue further extraction as described in 7.1. Ether 7.3 Fatty Crops, such as Oilseeds and Nuts Follow the extraction procedure described in 7.1. Evaporate chloroform completely on the water bath with the help of stream of air and dissolve the residue in 50 ml petroleum ether. Transfer the solution to a 250-ml separatory funnel and wash the beaker twice with 20 ml portions of petroleum ether trasferring the washings into the separatory funnel. Extract the solution in the separatory petroleum ether funnel thrice with 25-ml portions of acetonitrile. Pool the acetonitrile extract, and transfer to a second 250-ml separatory funnel and wash with 50"ml petroleum ether. Discard the petroleum ether layer. Transfer the acetonitrile solution directly to Ihe round bottom quantitatively, flask of the rotary vacuum evaporator and completely evaporate off the acentonitrile under slightly reduced pressure and with a water bath at 50°C. 7.4 Meat and Egg 1 N. Transfer a suitabl'e quantity ( 100-200 g > of the sample into a warinrr blender along with an equal qiantity of anhidrous sodiumYsclphate and blend the contents for 5 minutes. Add for 100 ml of chloroform, and homogenize further 5 minutes. Continue the extraction as per the procedure described in 7.1 and then continue as in 7.3. 7.5 Milk Transfer 200 ml of the milk sample into a 600-ml beaker, and ZOO-ml methanol and mix thoroughly by stirring with a glass rod. Introduce, with stirring, 20-ml sodium acetate buffer solution. Mix well and keep in the ice bath for Filter the mixture through a fluted 30 minutes. filter paper and rinse the filter with a jet of water. Collect the filtrate and water washings in a 600-ml beaker. Transfer the filtrate to a 1 OOO-ml separatory funnel and extract thrice with 60-ml portions of chloroform. Emulsions can be avoided by the addition of a 2-ml -saturated solution of sodium chloride. Pool the chloroform extracts in a 250-ml beaker and evaporate to dryness on a water bath with the help of a stream of air. 7.6 Water Transfer 500 ml of the water sample into a 1 OOO-ml separatory funnel and extract the aqueous layer thrice with 60-ml portions of chloroform. Pool the chloroform extracts in a 250-ml beaker and evaporate the contents to dryness on a water bath with the help of a stream of air. 2 + 6.3 Ethyl Ether tetrachloride. 5 percent - 6.4 Carbon Tetrachloride 6.5 Acetonitrile 6.6 Methanol - HPLC grade. HPLC grade. 6.7 Sodium Acetate Buffer Solution Mix equal volumes of 2 N acetic acid and 1 N sodiumhydroxide solution. 6.8 Reactivated Basic Alumina alumina with 10 ml water Mix 90 g basic thoroughly and allow to stand overnight. 6.9 Acetic Acid 2 N. ydroxide Solution 6.11 Sodium Sulpbate 6.12 Ethyl Acetate 6.13 Reference purity. 6.14 Reference purity. 7 EXTRACTION 7.1 Grain, Soil Straw, Hay Anhydrous. AR grade. Atrazine Simazine of known of known Standard Standard ( Low Moisture ) and Transfer a suitable quantity ( loo-209 g ) Gcely add ground sample into a Waring blender, and homogenize for 100-ml chloroform Trtinsfer the contents quantitatively 5 minutes. to a stoppered 1 000-ml conical flask, add 400-ml chloroform aLd shake the slurry vigorously on a rotary-action laboratory shaker for one hour. Filter the extract by decanting, through a layer of anhydrous sodium sulphate mounted on a Whatman No. 1 or equivalent filter paper and a funnel. Note the exact volume of the filtrate obtained. Evaporate an aliquot of the extract, equivalent to 50 g of the crop sample, carefully to dryness on a water bath in a 250-ml beaker, with the help of a gentle stream of air. 7.2 Fruit and Vegetables Transfer a suitable quantity ( 100 g ) of the finely chopped fruit or vegetable sample into a Waring blender along with about 100 g anhydrous sodium sulphate and 100 ml of chloroform. IS 13829 : 1993 8 CLEAN UP Add 25 g reactivated basic alumina to the chromatographic column, tap gently to eliminate channeling and to achieve uniform packing. Dissolve the extracted sample residue ( see 7 ) in lo-ml carbon tetrachloride, transfer on to the column and allow to penetrate into the alumina. Wash the beaker with 10 ml of carbon tetracolumn chloride and the transfer to This operation allowing to penetrate as before. shall be further repeated with another 5 ml of tetrachloride. When the solvent has just penetrated into the column, add SO ml of carbon tetlachloride and allow to pass through the alumina layer. When the last drop of the carbon tetrachloride has drained down, place a clean 250-ml beaker as receiver, and add 100 ml of 5 percent ethyl ether in carbon tetrachloride collecting the complete eluatc in the beaker. Evaporate the contents of the beaker to dryness on a water bath with a stream of air. 9 GAS CHROMATOGRAPHIC 9.1 Principle The residue of atrazine or simazine extracted from the sample after clean up is dissolved in ethyl acetate and estimated gas chromatographitally in an instrument equipped with a nitrogen specific detector. The content of atrazine or simazine in the sample is determined by comparing the response with the response of a known standard of similar concentration. 9.2 Prscedare Dissolve the residue after clean up ( see 8 ) in 2-ml ethyl acetate and inject 2 mlof this solution into the gas chromatograph. Identify the peak for atrazine or simazine by the retention time and measure $he peak area. 9.3 Calculation Residue of atrazine or simazine where A, = peak area of the sample; V, = volume, in ~1, of standard atrazine or simazine injected; v, = total volume, in ml, of the sample solutions; c = concentration, in tLg/g of the standard solution; f = recovery factor 100 = percent mean recovery ' es Al x V, x V, x c A2 x V, x M where A1 = peak area of the sample; v, = volume, v, = total C= As = peak area of the standard; V, = volume, in yl, of the sample injected; and M = mass, in g, of the sample taken for analysis. by NOTE - percent mean recovery is determined taking untreated control sample to which a known amount of atrazine or simazine is added and analyzed as described above. LIQUID PERFORMANCE 10 HIGH CHROMATOGRAPHIC ( HPLC > METHOD 10.1 Principle The residues of atrazine or simazine extracted from the sample after clean up is dissolved in methanol and estimated by HPLC equipped with ultra-violet detector. The content of atrazine or simazine is determined by comparing the response with the response of a known standard of similar concentration. 10.2 Procedure Dissolve the residue after clean u$ ( see 8 > in 2-ml methanol and inject. 5 ~1 of this solution Identify the peak into the I-IPLC instrument. for atrazine or simazine and measure the peak area. 10.3 Calculation Residue of atrazine or simazine ( pg/g > AI METHOD x AZ in ,ul, of standard atrazine or simazine injected; volume, in ml, of the sample solution; concentration, in pg/g, of the standard solutions; factor xf f= = recovery 100 percent mean recovery; in ~1, of the sample injected; and mass, in g, of the sample taken for analysis. AZ = peak area of the standard; v, = volume, M= by taking untreated control sample to which a known amount of atrazine or simazine is added and analyzed as described above. NOTE - Percent mean recovery is determined -Standard Mark Standards Act, 1986 and the Rules and Regulations by the provisions of the Bureau of Indian made thereunder. The Standard Mark on products covered by an Indian Standard conveys the assurance that they have been produced to comply with the requirements of that standard under a well defined system of inspection, testing and quality control which is devised and supervised by BIS and operated by the producer. Standard marked products are also continuously checked by BIS for conformity to that standard as a further safeguard. Details of conditions under which a icence for the use of the Standard Mark may be granted to manufacturers or producers may be obtained from the Bureau of Indian Standards. 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